T lymphocytes are responsible primarily for protection against intracellular pathogens and malignancies. Individuals who are grossly deficient in T-cell immunity frequently succumb to overwhelming infections by organisms such as cytomegalovirus, Pneumocystis carinii, Candida, and other apparently opportunistic pathogens, including bacteria, viruses, and fungi. These individuals may also succumb to malignancies such as B cell lymphomas, indicating the importance of T cell immunity in the suppression or elimination of certain tumors. Immunosuppression can result from a variety of causes, including viral infections (for example, with the HIV virus), as a result of chemical therapy, and malignancies (particularly of type that affect the hematopoietic system).
Mature T lymphocytes generally express the CD3 cell surface molecule, but consist predominantly of two basic subtypes based on their mutually exclusive expression of cell surface molecules CD4 and CD8. Most CD4+ T cells are involved in "helper" functions in immune responses and secrete cytokine molecules, in particular interleukin 2 (IL-2), upon which the cytotoxic CD8+ T cells are dependent. Such CD4+ T cells are often referred to as T helper (T.sub.H) cells. CD8+ cells are involved in "effector" functions in immune responses, such as direct cytotoxic destruction of target cells bearing foreign antigens, and represent an important mechanism for resistance to viral infections and tumors. The functional distinction between CD4+ and CD8+ T cells is based on the ability of CD4+ cells to recognize antigen presented in association with class II MHC molecules, and CD8+ cells to recognize antigen presented in association with class I MHC molecules. The CD8+ cells that mediate this lytic function are designated cytotoxic T lymphocytes (CTLs). Although most CTL are of the CD8+ phenotype, some CTL of the CD4+ phenotype have been described. Generally, individual CTLs (whether CD8+ or CD4+) are antigen-specific.
Lymphocytes are dependent upon a number of cytokines for proliferation. For example, CTLs are dependent on helper T (T.sub.H) cell-derived cytokines, such as IL-2, for growth and proliferation in response to foreign antigens. (Zinkernagel and Doherty, Adv. Immunol. 27:51, 1979; Male et al., Advanced Immunology, Chap. 7, Gower Publ., London, 1987; Jacobson et al., J. Immunol. 133:754, 1984). IL-2, for example, is a potent mitogen for cytotoxic T lymphocytes (Gillis and Smith, Nature 268:154, 1977), and the combination of antigen and IL-2 causes proliferation of primary CD8+ T cells in vitro. The importance of IL-2 for the growth and maintenance of the CD8+ CTL in vivo has been documented in models of adoptive immunotherapy in which the therapeutic efficacy of transferred anti-retroviral CD8+ cells is enhanced by subsequent administration of IL-2 (Cheever et al., J. Exp. Med. 155:968, 1982; Reddehase et al., J. Virol. 61:3102, 1987). IL-4 and IL-7 are also capable of stimulating the proliferation of at least a sub-population of mature CD8+ CTL (Alderson et al., J. Exp. Med. 172:577, 1990).
Considerable research has been focused on the use of T cells in treating malignant tumors and viral infections. Cytotoxic T cells specific for a particular type of tumor can be isolated and administered to a patient having a tumor with the effect that the CTLs ameliorate the tumor. It has been demonstrated, for example, that tumor-specific T cells can not only be generated to experimental tumors in mice but also that T cells with apparent tumor specificity can be isolated from human tumors. Such human tumor infiltrating lymphocytes (TILs) have been expanded in vitro and used to treat cancer patients, generating significant enthusiasm for human adoptive immunotherapy with tumor-specific T cells (Rosenberg et al., N. Engl. Med. 319:1767, 1988).
Similar studies using cytotoxic T cells specific for viral antigens have also been conducted in animal models. Human HIV-specific CTL of both the CD8+ (Walker et al., Nature 328:345, 1987; Plata et al., Nature 328:348, 1987) and CD4+ (Siliciano et al., Cell 54:561, 1988) phenotype have been isolated and characterized. HIV-specific CD8+ CTL are classical CTL in that their proliferative and cytotoxic responses are antigen-specific and MHC-restricted (Walker et al., supra; Chenciner et al., Eur. J. Immuno. 19:1537, 1989; Walker et al., Proc. Natl. Acad. Sci. USA 86:9514, 1989), in common with the numerous mouse and human CTL clones which have been characterized which are specific for viral, tumor or allospecific antigens.
The adoptive transfer of antigen (Ag)-specific T cells to establish immunity appears to be an effective therapy for some viral infections and tumors in the mouse animal model system (for a review see P. D. Greenberg, in Advances in Immunology, F. Dixon Ed. Academic Press, Inc. Orlando Fla. (1991), pp. 280-355). However, a successful outcome of an adoptive transfer method is dependent upon many factors, including the longevity of the transferred clones and the lack of toxicity to the host of the transferred cells. Although many antigen-specific T cell clones have been isolated, the use of tumor-specific T cell clones generated in vitro for tumor therapy has been shown to be subject to certain limitations. In particular, it has been demonstrated in several therapeutic models that the efficacy of cytolytic CD8+ T cells is limited by a dependency on exogenous IL-2 (produced by T.sub.H cells), a finding that has been substantiated in human adoptive therapy trials in which administration of exogenous IL-2 appears essential for optimal therapeutic efficacy (Rosenberg et al., N. Engl. J. Med. 319:1767, 1988; Klarnet et al., in Role of Interleukin-2 Activated Killer Cells in Cancer, Lutzova and Herberman (eds.), CRC Press, Florida, Chapter 14, pp. 199-218, 1990). Thus, while in vitro T cell cloning techniques provide a means to generate large numbers of T cells with demonstrable tumor or viral specificity, the full potential of using such antigen-specific T cells in therapy appears to be limited by their dependency on T.sub.H cells.
In some limited instances, the problem of T.sub.H dependency may be circumvented by using a particular class of CTLs that already function independently of T.sub.H cells. These cells are known as helper-independent cytolytic CD8+ cells (HIT.sub.c) (Klarnet et al., J. Immunol. 142:2187, 1989) and have been identified in some populations of primary (i.e., freshly isolated from in vivo sources) CD8+ CTL (Sprent and Schaefer, J. Exp. Med. 162:21068, 1985; Andrus et al., J. Exp. Med. 159:647, 1984). HIT.sub.c cells produce sufficient IL-2 to grow independently of CD4+ cells and the cytokines they produce. HIT.sub.c cells have been shown to express plasma membrane IL-1 receptors (IL-1R) and require IL-1 for their IL-2-independent proliferation (Klarnet et al., 1989, supra). This is in contrast to conventional CD8+ CTL which do not express detectable IL-1R on their surface (Lowenthal and MacDonald, 1987). HIT.sub.c cells have been generated which are specific for a range of antigens, including tumor, viral and alloantigens (von Boehmer et al., J. Immunol. 133:59, 1984; Klarnet et al., J. Immunol. 138:4012, 1987; and Andrus et al., J. Exp. Med. 149:647, 1984; Mizouchi et al., J. Immunol. 142:270, 1989). HIT.sub.c specific for a retrovirally transformed tumor have been shown to eradicate the tumor cells and persist long-term in vivo following their engraftment (Klarnet et al., 1989, supra). However, analogous human HIT.sub.c cells having specificity for many important antigens, such as HIV, have not yet been isolated.
Another significant limitation is associated with the phenomenon of T cell activation. In particular, activation of CD8+ T cells requires the interaction of multiple receptor-ligand pairs. Engagement of the T cell receptor (TCR) by antigen-MHC is one required interaction. However, it alone does not induce T cell activation but rather energy. Thus, "second signals" are believed to be essential for T cell activation (see, e.g., Weiss and Imboden, Adv. Immunol. 41:198, 1987; Weaver and Unanue, Immunol. Today, 11:49, 1990). These second signals are believed to be provided by interactions between co-stimulatory molecules present on the T cell and cognate ligands present on the surface of the antigen presenting cell (APC).
However, not all cells that are capable of presenting antigen will also possess the appropriate cognate ligands for co-stimulation. As a result, T cell activation may fail to occur because potential APCs that are encountered by the T cell may lack appropriate ligands, and, conversely, APCs having the cognate ligand may be relatively restricted in occurrence.
Realizing the full potential of antigen-specific T cells in therapy would be facilitated by developing a more complete repertoire of lymphocytes, in particular, CTLs that can be more readily activated and that have a lessened dependency on T.sub.H -cells. One approach, described by S. Lupton et al., has been to construct a hybrid gene comprising a region encoding a stimulatory factor polypeptide operably linked to a heterologous transcriptional control region, wherein the transcriptional control region causes activation-induced expression of the stimulatory factor encoding region when present in a lymphocyte that has been activated, and wherein expression of the stimulatory factor polypeptide in the activated lymphocyte reduces dependency of the lymphocyte on T helper cells (T.sub.H cells) (see U.S. Ser. No. 08/044,539, abandoned, filed 6 Apr. 1993; U.S. Ser. No. 08/244,448, U.S. Pat. No. 5,588,807, filed 6 Jun. 1994, and international counterpart PCT/US94/03679, filed 4 Apr. 1994). In that approach, activation of the lymphocyte results in coordinated expression of a stimulatory factor such as a cytokine.
In some situations, however, the lymphocytes may not be readily activatable. The present invention, described below, presents a different approach to the production of lymphocytes that are more readily activatable and that have a lessened dependency T helper cells.